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In situ Gelation Induced Death of Cancer Cells Based on Proteinosomes Yuting Zhou, Jianmin Song, Lei Wang, Xuting Xue, Xiaoman Liu, Hui Xie, and Xin Huang Biomacromolecules, Just Accepted Manuscript • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 21, 2017

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In situ Gelation Induced Death of Cancer Cells

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Based on Proteinosomes

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Yuting Zhou║, Jianmin Song║, Lei Wang*, Xuting Xue, Xiaoman Liu, Hui Xie* and Xin Huang* MIIT Key Laboratory of Critical Materials Technology for New Energy Conversion and Storage, State Key Laboratory of Robotics and Systems, School of Chemistry and Chemical Engineering, Harbin Institute of Technology, Harbin 150001, China. E-mails: [email protected], [email protected], [email protected]. ABSTRACT. Hydrogel is a nice kind of material, which can be utilized as a platform for cell culture. However, when bulky hydrogel was formed inside cancer cells, results would be changed. In this study, we showed a way of in situ gelation inside the cancer cells which could efficiently induce the death of cancer cells. Glutathione (GSH) responsive proteinosomes, with good biocompatibility, were prepared as carriers for sodium alginate to be endocytosed by cancer cells, where the chelation between sodium alginate and free calcium ions in the culture medium occurred during the diffusion process. The uptake of the hydrogel-loaded proteinosomes into the cancer cells, and then the triggered release of hydrogel with concomitant aggregation, was well confirmed by monitoring the change of the Young’s modulus of the cells based on AFM force measurement. Accordingly, when a large amount of hydrogel formed in cells, the cell viability would be inhibited about 90% at the concentration of 5.0 µM of hydrogel-loaded proteinosomes after 48 h incubation by MTT assay, which clearly proved the feasibility of the demonstrated way to kill cancer cells. Although more details about the mechanism of cell death should be conducted in the near future, such demonstrated way of in situ gelation inside cells would contribute another choice for killing cancer cells. KEY WORDS. Proteinosomes, alginate hydrogel, in situ gelation, AFM force measurement, redox response INTRODUCTION Cancer is the leading disease of mortality in many countries around the world.1 Therefore, how to develop a safe and effective treatment for cancer has become the research emphasis for modern science and technology.2-6 Among various mainstream cancer therapies (e.g. chemotherapy, radiotherapy), limited therapeutic efficacy has been revealed owing to poor bioavailability, and likely to induce multi-drug resistance (MDR).7-10 In recent years, some other methods have been discovered to resolve the aforementioned problems, including bacteria therapy,11, 12 gene therapy13, 14 and ion therapy15, 16. However, the current mortality rate caused by cancer still remains high, 1 ACS Paragon Plus Environment

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which is mainly because of the inability to deliver therapeutic agents only and directly to the tumor sites, without inducing severe adverse effects on healthy tissues and organs. Therefore, it would be desirable to develop highly efficient therapeutics, which have excellent biocompatibility, and can selectively respond to the heterogeneous and complex microenvironment inside a tumor for on-demand release of therapeutic agents, or directly generate intracellular self-assembly to cause the death of cells. To address the above problems, on the one hand, the delivery of anticancer drugs based on micro/nano carriers become the research focus with many attractive features, including better protection of drugs, targeted delivery and stimuli-responsive controlled release.17 Thus, many different materials were employed as drug carriers, including various inorganic nanoparticles18, protein conjugates19, liposomes20, polymersomes3 and hydrogels21, to name a few. Amongst these carriers, protein-based capsules can offer significant advantages over purely synthetic systems, and the use of proteins as the precursor materials often results in the minimal immunogenicity.22 As a representative protein, albumin is non-toxic, water-soluble, biodegradable and most abundant in human plasma; in the meanwhile, it is also stable over a large pH range of 4 to 9 and relatively high temperature of 60 °C, thus making it an ideal material for the preparation of protein-based drug carriers.23-25 For example, Zare et al. reported BSA-PMMA nanoparticles loaded with camptothecin exhibited enhanced anti-tumor activity both in vitro and in vivo.26 Stenzel et al. investigated the influence of the albumin content conjugated on the surface of polymer nanoparticles on the accumulation and the subsequent cytotoxicity to different cell lines, which offered better understanding of the effect of albumin on cancer cells27, 28, and further stimulated the development of protein-based carriers. In this regard, recently, we demonstrated a way to generate protein-polymer based capsule, i.e. proteinosome, the biocompatible microscale compartments delineated by a semi-permeable membrane comprising of a closely packed monolayer of conjugated protein-polymer building blocks, with the great potential for drug delivery in its hollow inner cavity.29 On the other hand, the generation of intracellular self-assembly to cause the death of cells would be an excellent anticancer therapy, due to the high efficiency. Xu et al. reported the pioneering work that synthetic organic molecules, which penetrated cell membranes and self-assembled to construct nanostructures inside the living cells, significantly affected cellular function.30 This idea was further demonstrated by using the enzyme-instructed or cellular specific expression triggered intracellular self-assembly of a peptide lipid31, aromatic carbohydrate amphiphile32, or redox modulators33, etc. These work offered new insights into intracellular anti-cancer methods. Atomic force microscopy technique (AFM) has been used incrementally over the past decade in cell biology.34 Besides its usefulness in high resolution imaging, AFM also has unique capabilities for probing the viscoelastic properties of living cells in culture, thus providing an indirect indicator of the structure and function of the underlying cytoskeleton and cell organelles.35 Furthermore, AFM measurements have 2 ACS Paragon Plus Environment

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boosted the understanding of cell mechanics in normal and diseased states, and provide the potential novel diagnostic and treatment options. Besides, as we know, hydrogel can be utilized as drug delivery materials and cell culture media, due to its capability of retaining water or physiological fluids in large quantities, thus reducing interfacial tension with biological fluids and promoting biocompatibility. Among various hydrogels, sodium alginate typically exhibits good biocompatibility and biodegradability, which can chelate calcium ions to generate hydrogel,36, 37 however, when the amount and place changed, the effect would also change. Herein, we designed and constructed a GSH-responsive proteinosome-based carrier to demonstrate that in situ intracellular gelation of alginate hydrogel could induce the death of cancer cells, which showed a promising application as a potential concept of gel therapy. This work has several advantages: i) the synthesized small size glutathione (GSH)-responsive BSA-NH2/PNIPAAm proteinosomes demonstrated high loading efficiency of sodium alginate as well as nice biocompatibility towards the used cancer cells;29, 38, 39 ii) high concentration of GSH in cancer cells can break the disulfide bonds of the crosslinker (NHS-PEG16-DS disulfide ester) of proteinosomes, thus leading to the in situ intracellular gelation of cytolymph;40, 41 iii) AFM force measurement was specially employed to monitor the change of the Young’s modulus of cancer cells, which well confirmed the gelation process inside the cancer cells and the induced death of the cells.42, 43 This work will further demonstrate the intracellular reaction induced cell death, with the great potential for future clinical applications. EXPERIMENTAL SECTION Materials. N-isopropylacrylamide (NIPAAm, 98%) was recrystallized twice in hexane and toluene before use. NHS-PEG16-DS disulfide ester (4, 7, 10, 13, 16, 19, 22, 25, 32, 35, 38, 41, 44, 47, 50, 53-hexadecaoxa-28,29-dithiahexapentacontanedioic acid di-N-succinimidyl ester) (MW, 1109 Da, ≥98%), 1,6-diaminohexane (≥98%), 2-ethyl-1-hexanol (≥98%), calcium chloride (CaCl2), sodium alginate (NaAlg), glutathione (GSH), 2,4,6-trinitrobenzene sulfonic acid (TNBSA), p-nitrophenylacetate, albumin from bovine serum (BSA) (≥98%, Mw 66 kDa), fluorescein isothiocyanate labelled dextran (FITC-Dextran, Mw 150 kDa) and N-ethyl-N’(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) (98%) were purchased from Sigma Aldrich, China. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4.00 mM glutamine, 4500 mg/L glucose and Phosphate Buffered Saline (PBS) were acquired from HyClone Laboratories Co., Ltd., (Logan, Utah, USA) and 10% (v/v) fetal bovine serum (FBS) was purchased from Solarbio Life Sciences Co., Ltd. (Beijing, China). Characterizations. Transmission electron microscopy (TEM). TEM analysis was undertaken using a JEM-1400 and a LaB6 filament at 120 kV in bright field mode. Samples were prepared by adding one drop of proteinosome solution (0.1 mg/mL) onto a 300-mesh carbon film coated copper grid and the specimens were then dried in vacuum for one day. Scanning electron microscopy (SEM). SEM images were obtained on a Hitachi FE-SEM SU8000. Optical and fluorescence microscopy were 3 ACS Paragon Plus Environment

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performed on a Leica DMI8 manual inverted fluorescence microscope at 10x, 20x, 40x and 100x magnifications. Dynamic light scattering (DLS). The size distribution of the capsules was characterized using DLS with an ALV-5000/E DLS instrument (Malvern Instruments, UK), with a fixed scattering angle of 90°. The pH values were measured with a SevenCompact meter (METTLER TOLEDO, SUI). Sonication process was conducted in a SB-5200 DTDN ultrasonic device. Silicon nitride rectangular cantilevers with V-shaped tip (100 µm long, 30 µm wide and 0.13 µm thick) utilized in the AFM experiments was from ‘Bio-Lever series’ (BL-RC150VB-C1, Olympus Co., Japan). 1H NMR spectra were recorded using a Jeol Lambda 300 spectrometer operating at 300 MHz (1H), with CDCl3 as solvents at room temperature. Preparation of Cationized Bovine Serum Albumin (BSA-NH2). BSA-NH2 was synthesized by carbodiimide activated conjugation of 1,6-diaminohexane to aspartic and glutamic acid residues on the external surface of the protein. For details, a solution of 1,6-diaminohexane (1.5 g) was adjusted to pH of 6.5 using 5 M HCl, and added dropwise to a stirred solution of BSA (200 mg in 50 mL of water). The coupling reaction was initiated by the immediate addition of N-ethyl-N’-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC, 100 mg in 1 mL of water), and again (50 mg in 1 mL of water) after 5 h. The pH value was maintained at 6.5 with the adjustment of using dilute HCl, and the solution was stirred for a further 6 h. The solution was then centrifuged to remove any precipitate and the supernatant was dialyzed (dialysis tubing 12-14 kDa MWCO) extensively against Milli-Q water. The final product was obtained by freeze-drying. Nanoconjugates.29 End-capped Preparation of BSA-NH2/PNIPAAm mercaptothi- azoline-activated PNIPAAm (Mn 15,000 g/mol, 10 mg in 5 mL of water, 1 H NMR was shown as Fig. S1) 29 was added to a stirred solution of BSA-NH2 (10 mg, in 5 mL of PBS buffer pH 8.0). The mixed solution was stirred for 12 h, and then purified by using a centrifugal filter (Mw 50 kDa) to remove any unreacted PNIPAAm and salts. After freeze-drying, the BSA-NH2/PNIPAAm conjugate was obtained, with the molecular weight of ca. 97 kDa. Preparation of Proteinosomes. Proteinosomes were prepared by mixing an aqueous BSA-NH2/PNIPAAm solution with 2-ethyl-1-hexanol followed by sonication (4 min, 300 W). The samples were prepared at a constant aqueous/oil volume fraction of 0.06. Typically, 0.06 mL of aqueous BSA-NH2/PNIPAAm (20.0 mg/mL, pH 8.5, sodium carbonate buffer) were mixed with 1.0 mL of the oil. The proteinosomes were then crosslinked by addition of NHS-PEG16-DS disulfide ester (0.5 mg) into water phase, which reacted with free primary amine groups of BSA-NH2. The transfer of cross-linked proteinosomes into water was achieved as follows: after 12 h sedimentation, the upper clear oil layer was discarded, and 0.5 mL of 70 % ethanol added and the emulsion gently shaken. The dispersion was dialyzed against 70 % ethanol/water for 3 h, and then the solution was dispersed into 20 mL of Milli-Q water. For the encapsulation, NaAlg (0.2 mg) or/and FITC-Dextran (0.05 mg) were added 4 ACS Paragon Plus Environment

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into the aqueous BSA-NH2/PNIPAAm solution and mixed uniformly, followed by mixing with the oil phase to obtain the proteinosomes loaded with NaAlg or/and FITC-Dextran. Cell Culture. HepG2 cells and NIH 3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 4 mM glutamine, 4500 mg/L glucose and 10 % (v/v) fetal bovine serum (FBS). The cells were incubated at 37 ℃ under a humidified atmosphere containing 95 % air and 5 % CO2. When doing the gel therapy, CaCl2 solution was added in to the medium, with an optimized concentration of 1.5 mg/mL. Cellular Uptake of Proteinosomes. The concentration-dependent cellular uptake behaviors of HepG2 cells were studied by fluorescence microscopy. HepG2 cells were seeded on 35 mm dishes in 1 mL of DMEM supplemented with 4 mM glutamine, 4500 mg/L glucose and 10 % (v/v) fetal bovine serum (FBS). After HepG2 cells were completely adhered, FITC-Dextran-loaded proteinosomes at different concentrations of 0.3125, 0.625, 1.25, 2.5 and 5.0 µM was added to different dishes and incubated at 37 ℃ for 36 h, respectively. Thereafter, culture medium was removed and cells were washed with PBS for three times. The resulting dishes were mounted and observed with a Leica DMI8 manual inverted fluorescence microscope. Atomic Force Microscope (AFM) Measurement. In this study, we proposed an independent development AFM-based nanorobotics system for the cell force measurement.44 The half-opening angle of the tip apex was 45°. The spring constant of the cantilevers was determined using the method proposed by Sader et. al.45, with values of 0.006 N/m. The relationship between force and deformation of the AFM probe is in accordance with Hooke’s law.46 That is, when the cantilever was exerted a small force, a suitable deformation could be detected. Meanwhile, the probe stiffness need to be close to the stiffness of samples. When the deformation of the probe caused by the force is basically the same as the deformation of the sample itself, the surface topography and mechanical properties of the sample are most similar to the actual situation.47 Optimization of the Experimental Parameter of AFM. In view of the fluid resistance in the liquid environment (PBS solution, pH 7.4) and the small Young’s modulus, cell mechanics measurements in liquid were performed using a one-step manual measurement, and the Young’s modulus was calculated from the data curve. During the measurement, the moving speed of the probe was 400 nm/s to carry out the pull-off test. Each pull-off test interval was 4 s to ensure that the cells had sufficient time to recover the deformation of the last pull-off test, thus ensuring that each measurement data was as close as the actual data.48, 49 Calculation Model of the AFM Data. The cell deformation conformed to the modified Hertz model.50, 51 Because the loading rate was low and the cell deformation was less than half of the cell height. The apparent Young’s modulus E of cells was related to the position of the contact point between cell surface, the tip and the slope of the force-distance curve. The modified Hertz model for a stiff cone and a flat 5 ACS Paragon Plus Environment

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1

surface can be expressed as:

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where, F indicates the force of probe, v represents Poisson’s ratio of cells of 0.5 in this system52, δ is the indentation depth of the tip. The point-to-point search method is used to find the contact point between the cell and the tip by Matlab. The force-distance curve behind the contact point can be used to calculate the slope, and finally Young’s modulus can be obtained. MTT Assay. The cell viability of human HepG2 cells and NIH 3T3 cells in presence of NaAlg-loaded proteinosomes with the addition of Ca2+ ions was determined using MTT method.53 The NaAlg-loaded proteinosomes without Ca2+ ions and proteinosomes containing PBS were used as control. Cells were seeded in a 96-well plate with a density of 1.0×104 cells per well in 150 µL of culture medium. After 12 h incubation, the medium was replaced with 150 µL of medium containing serial dilutions of NaAlg-loaded proteinosomes with and without the addition of Ca2+ ions, and proteinosomes containing PBS from 0.3125 to 5.0 µM, respectively. The cells were incubated (37 ℃, 5 % CO2) for another 24, 36 and 48h, respectively. Then the medium was replaced with a solution of 3-(4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl-2H-tetrazolium bromide (MTT) in medium (100 µL, 1.0 mg/mL),11 followed by 4 h incubation. The medium was then replaced by DMSO (100 µL/well). The absorbance was measured in a Microplate Spectrophotometer at a wavelength of 490 nm. The blank was subtracted to the measured optical density (OD) values, and the cell viability was normalized to the absorbance measured from the untreated control cells.

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Construction of GSH-responsive Proteinosome-Based Microcarriers. The general procedure of the designed in situ gelation induced cancer cell death was described in Scheme 1. Basically, a dispersion of proteinosomes was obtained by sonicating the mixture of water phase containing BSA-NH2/PNIPAAm (BSA-P, Scheme 1a), NaAlg and oil phase of 2-ethyl-1-hexanol (Scheme 1b). Proteinosomes loaded with NaAlg was crosslinked, followed by the removal of oil phase by dialysis against water/ethanol mixture to transfer the microcapsules into a continuous water phase (Scheme 1c). In the presence of Ca2+ ions in the culture medium, the hydrogel could form inside the proteinosomes by the chelation between the loaded NaAlg and Ca2+ ions, and then the proteinosomes could enter tumor cells by endocytosis (Scheme 1d). High concentration of glutathione (GSH) in tumor cells would break disulfide bonds (S-S) in the crosslinker (Scheme 1e), causing the disassembly of proteinosomes membrane and the release of hydrogel inside the cells. Subsequently, the bulky lumps formed by the aggregation of released hydrogel (Scheme 1e) which would inhibit the flowing and communication of cytoplasm, thus inducing the death of cancer cells.54 6 ACS Paragon Plus Environment

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Scheme 1. Schematic illustration showing the procedure of the fabrication of proteinosomes containing sodium alginate (NaAlg) and the in situ gelation inside cancer cells. (a) Preparation of BSA-NH2/PNIPAAm nanoconjugates based on the reaction between the amino groups (-NH2) on the external surface of BSA and mercaptothiazoline groups of PNIPAAm. (b) Preparation of proteinosomes containing NaAlg by sonication. (c) The proteinosomes were crosslinked using a disulfide-containing crosslinker and transferred to the water phase. (d) NaAlg chelated with free Ca2+ ions in the culture medium to form hydrogel inside proteinosomes. (e) Glutathione (GSH) in the cancer cell cleaved disulfide bonds in the crosslinker, leading to the in situ formation of bulky hydrogel which induced the death of cancer cells.

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Fig. 1 Optical (a) and fluorescence (b) microscopy images of proteinosomes in oil; the fluorescence originates from encapsulated fluorescein isothiocyanate labeled dextran (FITC-Dextran). (c) Optical microscopy images of proteinosomes dispersed in aqueous solution. (d) The dynamic light scattering (DLS) plot of proteinosomes dispersed in aqueous solution. Optical (e), fluorescence (f), TEM (g) and SEM (h) images of proteinosomes after drying in air. To fabricate the microcarriers of proteinosomes, the building blocks of amphiphilic BSA-P conjugates were synthesized and characterized according to our previous report.29 Then, an aqueous solution of BSA-P and 2-ethyl-1-hexanol was mixed at an aqueous/oil volume fraction of 0.06, and sonicated to produce proteinosomes via the spontaneous assembly of amphiphilic building blocks at the interface of water droplets in oil. They were in the form of hollow spherical microstructures (Fig. 1a), with loaded hydrophilic fluorescence dye (FITC-Dextran) (Fig. 1b) inside the water phase, and hydrophobic fluorescence dye (Nile Red) in the oil phase (Fig. S4). After crosslinking and dialyzing against ethanol/water mixture, the proteinosomes could be transferred into aqueous phase (Fig. 1c) with an average diameter of 1.32 µm from the DLS measurement. Proteinosomes were further characterized using optical (Fig.1e), FL microscopy (Fig. 1f), TEM (Fig. 1g), SEM (Fig. 1h) and AFM (Fig. S5), from which collapsed but intact structures with continuous and flexible membranes were observed. Additionally, the average diameter of proteinosomes could be systematically controlled by tuning the concentration of BSA-P conjugates, sonication time and sonication power (Fig. S6-8). Basically, the increase of the concentration of 8 ACS Paragon Plus Environment

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the BSA-P building blocks from 5 to 20 mg/ mL resulted in the decrease of the mean size of proteinosomes (Fig. S6), with no more change when the concentration increased to 25 mg/mL (Fig. S3f). Thus, the concentration of 20 mg/mL was chosen for the preparation of proteinosomes. Similarly, sonication time and power were also optimized (Fig. S7-8), with the conclusion that longer sonication time and higher sonication power lead to the generation of smaller proteinosomes. Accordingly, in the following study, the sonication time of 4 min and sonication power of 300 W were employed to prepare proteinosomes, with the size around 1 µm (Fig. S4-5), since this was a suitable size for proteinosomes which can be endocytosed by cells in the following study.55 Additionally, the activity of BSA was evaluated under this condition (Fig. S9), which showed the sonication process had little effect on the activity of BSA. In order to transfer the proteinosomes into pure water phase, the concentration of crosslinker was also optimized (Fig. S10), with the optimal concentration of 12.5 mg/mL. The temperature effect on the formation of proteinosomes containing NaAlg was also investigated (Fig. S11-12). From the results, it was easy to see that the temperature had little impact on the formation and the stability of the proteinosomes in our studied temperature range. GSH Triggered Release of Hydrogel and Concomitant Aggregation in vitro. In order to demonstrate the formation of alginate hydrogel inside proteinosomes and then the GSH triggered release of the hydrogel with concomitant aggregation, large proteinosomes with the size ca. 20 µm were prepared. Firstly, NaAlg loaded proteinosomes, crosslinked by disulfide-containing cross-linker, were synthesized and transferred into water phase based on the aforementioned conditions (Fig. 2a). From the negative phase contrast image, it was hard to see the contrast difference of proteinosomes (Fig. 2b), since both inside and outside proteinosomes were aqueous phase. While 10 min later after adding CaCl2 solution into the system (concentration optimization shown in Fig. S13-14), hydrogel formed inside the proteinosomes due to the chelation between NaAlg and free Ca2+ ions, as confirmed by the appearance of the unsmooth and rugged membrane of proteinosomes (Fig. 2c), as well as the increased contrast and the maintenance of the intact sphere structures without collapsing (Fig. 2d, Fig. S15). Then, when GSH (2 mM) was added into the solution, the membrane was broken due to the cleavage of the disulfide bonds of the cross-linkers in the membrane, leading to the aggregation of released bare hydrogels into bulky lumps after ca. 4 h (Fig. 2e, f).40 This was also clearly observed from the change of proteinosomes in bulk solution (insets of Fig. 2). The solution containing proteinosomes was clear and transparent initially (Fig. 2a, inset), and changed into blurred milk color (Fig. 2c, inset) with the addition of CaCl2 solution, again indicating the formation of the hydrogel. At last, when GSH was added into the solution, the GSH-triggered release of the hydrogel and the concomitant aggregation was confirmed by the appearance of the macroscopic flocculation at the bottom of the tube (Fig. 2e, inset). Moreover, the release percentage was calculated to be 92.7 % under the concentration of GSH 2.0 mM in the studied system (Fig. S16-17). 9 ACS Paragon Plus Environment

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Fig. 2 (a, c, e) Optical microscopy and (b, d, f) phase contrast microscopy images of large proteinosomes dispersed in aqueous solution under different conditions showing the formation of alginate hydrogel inside proteinosomes, and then the GSH triggered release of the hydrogel with the concomitant aggregation: (a, b) proteinosomes containing sodium alginate; (c, d) after adding Ca2+ ions to (a); (e, f) after adding GSH to (c). The insets are photos of proteinosomes dispersed in aqueous solution, where the red circles showed the location of proteinosomes under different situations. Biocompatibility and Cellular Uptake of Proteinosomes. Subsequently, the biocompatibility of proteinosomes was investigated by using human hepatocellular liver carcinoma cell line (HepG2 cell) and NIH 3T3 cells. The concentration-dependent cellular uptake behaviors were studied in detail by incubating HepG2 cells and NIH 3T3 cells with FITC-Dextran (Mw 150 kDa) loaded proteinosomes under different concentrations from 0.3125, 0.625, 1.25, 2.5, to 5.0 µM for 36 h, respectively (Fig. S18-19). The uptake amount increased obviously with the growth of the concentration of proteinosomes as seen from the FL microscopy images. When the concentration was more than 2.5 µM, the areas occupied by green color in cells did not increase any more (Fig. S18d, e), indicating that the uptake amount reached the plateau. Accordingly, the concentration of proteinosomes for the following study was set to be 2.5 µM. Furthermore, FL images of HepG2 cells incubating with FITC-dextran-loaded proteinosomes at different times were shown in Fig. 3. By the comparison of the images after 24 h (Fig. 3g,h) and 36 h (Fig. 3k,l) incubation, there was an obvious increase on the green fluorescence area inside the cells after extending the incubation time to be 36 h. Considering that the excessive FITC-dextran-loaded proteinosomes in the culture medium had be removed after 24 h incubation, obviously, this was due to the fact that GSH inside HepG2 cells triggered the release of the loaded FITC-dextran in the proteinosomes by breaking disulfide bonds in the membrane of the proteinosomes. 10 ACS Paragon Plus Environment

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Fig. 3 The schematic (a, e, i), optical (b, f, j), FL (c, g, k) and merged (d, h, l) images showing cancer cells (HepG2 cells) incubated without proteinosomes (a-d), and with FITC-dextran loaded proteinosomes for 24 h (e-h) and 36 h (i-l). AFM Force Measurement for Young’s Modulus (EY) of Cancer Cells. To further confirm the endocytosis of the proteinosomes by HepG2 cells, the AFM force measurement was employed by monitoring the Young’s modulus (EY) of the cancer cells during the whole process. Since EY can give a good reflection of the status of live cells, especially when different materials were endocytosed. To confirm so, first, normal cancer cells were tested, illustrated in Fig. 4a inset. From the results, it was obvious to see EY of cancer cells was in the range from 400 to 1000 Pa (Fig. 4a), which was consistent with previous results48. This result also suggested that the AFM probe and the calculation based on Hertz model in this system were appropriate, thus could be utilized for further characterizations. As a control experiment, cancer cells endocytosed pure proteinosomes containing PBS solution were tested after incubation for 24 h, with EY values ranging from 400 to 2400 Pa (Fig. 4b). This increase was caused by the uptake of proteinosomes, since proteinosomes were relatively rigid compared with pure cell membrane.56 Significantly, after incubating the same sample for 36 h, EY values recovered (Fig. 4c), which well suggested that the endocytosed proteinosomes were digested and lost the rigid capsule structure, and this was consistent with our analysis from the fluorescence images (Fig. 3e-l).

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Fig. 4 The statistical histograms of Young’s modulus measured by AFM under different conditions: normal cancer cells (a), cancer cells endocytosed pure proteinosomes for 24 h (b) and 36 h (c); cancer cells endocytosed proteinosomes containing NaAlg for 24 h (d), 36 h (e) and 48 h (f). Insets are the schematic illustration of different test samples (tested cell number >100). Based on the above observations, subsequently the NaAlg-loaded proteinosomes, after being endocytosed, its release of loaded hydrogel and the concomitant aggregation in HepG2 cells were investigated. After 24 h incubation, the endocytosis of the hydrogel-filled proteinosomes by HepG2 cells was tested. The EY histogram appeared bimodal distribution, with one ranging from 400 to 2400 Pa and the other ranging from 6500 to 9500 Pa (Fig. 4d). The lower peak was attributed to the normal area of cancer cells, and the higher peak was due to the proteinosomes with formed hydrogel inside, since the control experiment showed that EY of proteinosomes containing hydrogel was in the range of 6000-10000 Pa (Fig. S22), which further proved the successful uptake of hydrogel-filled proteinosomes. After incubation for 36 h (Fig. 4e), the left peak ranging from 300 to 1300 Pa decreased, while the right peak increased, suggesting that the diffusion and spread of aggregated hydrogel inside cells leading to the decline of low EY percentage. While, after 48 h incubation, the left peak disappeared, and the right peak’s width-at-half-height increased further (Fig. 4f), indicating that the hydrogel kept releasing and aggregating inside cells, and occupied most area of cell cytolymph which were also consistent with the studied fluorescence experiments. 12 ACS Paragon Plus Environment

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Fig. 5 MTT toxicity assay with HepG2 and NIH 3T3 cells. Inhibitory effect of proteinosomes in PBS solution, proteinosomes-NaAlg, proteinosomes-CaAlg within HepG2 cells and proteinosomes-CaAlg within NIH 3T3 cells, after 24 h (a), 36 h (b) and 48 h (c) (n = 3, error bar = S.D.).

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MTT Assay. For such HepG2 cells with formed bulky hydrogel in the cytolymph, their viability was evaluated by using 3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide (MTT) assay, with NIH 3T3 cells as controls. As shown in Figure 5, both proteinosomes containing PBS solution and proteinosomes-NaAlg exhibited very low cytotoxicity against HepG2 cells after 24, 36 and 48 h incubation. For example, after 48 h incubation, proteinosomes containing PBS solution and proteinosomes-NaAlg caused less than 20% reduction in viability compared to untreated cells even when the concentration of proteinosomes containing PBS solution and proteinosomes-NaAlg were up to 5.0 µM. While, for the hydrogel-filled proteinosomes (proteinosomes-CaAlg), after 24 h incubation, less than 10% reduction in viability caused by proteinosomes-CaAlg at the concentration of 1.25 µM (Fig. 5b), which indicated that little amount of hydrogel inside cells had little inhibited effect. Thereafter, if extended the incubation time to be 36 h, the viability of cancer cells started to decrease, which was consistent with the fluorescence and AFM results, also indicating the release of hydrogel and the concomitant aggregation. Moreover, the studied cell viability percentage could be decreased further to be 20% and 10% when the cells were incubated with 2.5 and 5.0 µM of hydrogel-filled proteinosomes after 48 h, respectively, compared with those of 92% and 89% under the same condition but without forming the hydrogel. Compared with the viability of NIH 3T3 cells (Fig. S23), the inhibited effect of proteinosome, proteinosomes containing NaAlg, proteinosomes containing CaAlg was much lower. Therefore, these findings could verify that the in situ gelation inside cancer cells could affect the viability dramatically. CONCLUSIONS In this study, we should successfully demonstrate the construction of a type of GSHresponsive sodium alginate loaded BSA-NH2/PNIPAAm proteinosomes with excellent biocompatibility, of which the average diameter could be well controlled by combined regulation of the concentration of BSA-NH2/PNIPAAm conjugates, sonication time and sonication power. The ca. 1 µm of the constructed calcium alginate-hydrogel loaded proteinosomes could be endocytosed by HepG2 cells 13 ACS Paragon Plus Environment

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efficiently, with the triggered release of the loaded hydrogel via the cleaving of disulfide bonds caused by the reaction between GSH in the cell cytoplasm and the crosslinker in the membrane of the proteinosomes. Significantly, the released hydrogel could aggregate into bulky lumps, which then would affect the cell viability dramatically. The whole procedure was well demonstrated by monitoring the change of the Young’s modulus of cancer cells, and the corresponding cell viability was confirmed by MTT assay, using NIH 3T3 cells as controls. Overall, it is anticipated that the demonstrated in situ gelation inside the cancer cells could open a new way for the future cancer therapy with the potential to be applied to clinical areas. ASSOCIATED CONTENT Supporting Information The supporting information is available free of charge on the ACS Publication website at DOI:10.1021/XXX. Additional characterization data for BSA-P, optimization of fabrication conditions, details of the gel formation conditions, the images for in vitro results, and details for the AFM force measurements. AUTHOR INFORMATION Corresponding Authors *E-mails: [email protected]; [email protected]; [email protected] Notes ║These authors contributed equally to this work. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank NSFC (Nos. 51521003, 61573121 and 21474025), China Postdoctoral Science Foundation (2016M600247, 2015M571401), Fundamental Research Funds for the Central Universities (HIT.BRETIV.201309), and Postdoctoral Science Foundation of Heilongjiang Province (LBH-Z16066, L. W.). REFERENCES 1. Jemal, A.; Bray, F.; Center, M. M.; Ferlay, J.; Ward, E.; Forman, D. CA Cancer J. Clin. 2011, 61, (2), 69-90. 2. Tucker, B. S.; Getchell, S. G.; Hill, M. R.; Sumerlin, B. S. Polym. Chem. 2015, 6, (23), 4258-4263. 3. Huo, M.; Yuan, J.; Tao, L.; Wei, Y. Polym. Chem. 2014, 5, (5), 1519-1528. 4. Zhao, J.; Lai, H.; Lu, H.; Barnerkowollik, C.; Stenzel, M. H.; Xiao, P. Biomacromolecules 2016, 17, (9), 2946-2955. 5. Wang, N.; Jin, X.; Guo, D.; Tong, G.; Zhu, X. Biomacromolecules 2016, 461-474. 6. Chen, W.; Zou, Y.; Meng, F.; Cheng, R.; Deng, C.; Feijen, J.; Zhong, Z. Biomacromolecules 2014, 15, (3), 900-907. 7. Yang, G.; Liu, J.; Wu, Y.; Feng, L.; Liu, Z. Coordin. Chem. Rev. 2016, 320, 100-117. 8. Xin, Y.; Huang, Q.; Tang, J.-Q.; Hou, X.-Y.; Zhang, P.; Zhang, L. Z.; Jiang, G. Cancer Lett. 2016, 379, (1), 24-31. 9. He, Q.; Shi, J. Adv. Mater. 2014, 26, (3), 391-411.

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Fig. 1 Optical (a) and fluorescence (b) microscopy images of proteinosomes in oil; the fluorescence originates from encapsulated fluorescein isothiocyanate labeled dextran (FITC-Dextran). (c) Optical microscopy images of proteinosomes dispersed in aqueous solution. (d) The dynamic light scattering (DLS) plot of proteinosomes dispersed in aqueous solution. Optical (e), fluorescence (f), TEM (g) and SEM (h) images of proteinosomes after drying in air. 153x228mm (100 x 96 DPI)

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Fig. 2 (a, c, e) Optical microscopy and (b, d, f) phase contrast microscopy images of large proteinosomes dispersed in aqueous solution, under different conditions showing the formation of alginate hydrogel inside proteinosomes and then the GSH triggered release of the hydrogel with the concomitant aggregation: (a, b) proteinosomes containing sodium alginate; (c, d) after adding Ca2+ ions to (a); (e, f) after adding GSH to (c). The insets are photos of proteinosomes dispersed in aqueous solution, where the red circles emphasized the location of proteinosomes under different situations. 146x171mm (150 x 150 DPI)

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Fig. 3 The schematic (a, e, i), optical (b, f, j), FL (c, g, k) and merged (d, h, l) images showing cancer cells (HepG2 cells) incubated without proteinosomes (a-d), and with FITC-dextran loaded proteinosomes for 24 h (e-h) and 36 h (i-l). 302x183mm (136 x 136 DPI)

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Fig. 4 The statistical histograms of Young’s modulus measured by AFM under different conditions: normal cancer cells (a), cancer cells endocytosed pure Proteinosomes for 24 h (b) and 36 h (c); cancer cells endocytosed proteinosomes containing NaAlg for 24 h (d), 36 h (e) and 48 h (f). Insets are the schematic illustration of different test samples (tested cell number >100). 112x83mm (300 x 300 DPI)

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Fig. 5 MTT toxicity assay with HepG2 and NIH 3T3 cells. Inhibitory effect of proteinosomes in PBS solution, proteinosomes-NaAlg, proteinosomes-CaAlg within HepG2 cells and proteinosomes-CaAlg within NIH 3T3 cells, after 24 h (a), 36 h (b) and 48 h (c) (n = 3, error bar = S.D.). 253x76mm (150 x 150 DPI)

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